Overview

Over the last century, antimicrobials have been used to treat bacterial, fungal, and some parasitic infections. However, misuse has led to antimicrobial resistance (AMR), making infections harder to treat. Current efforts to develop new antimicrobials are both time consuming and costly (WHO, 2023). Lambert iGEM proposes using CRISPR-interference (CRISPRi) as a novel approach to combat bacterial diseases. In conjunction with this system, our team developed a toehold RNA system for the detection of target genes in order to validate the efficacy of our CRISRPi mechanism.

Justification

While evaluating CRISPRi’s potential as a therapeutic, a problem arose when considering testing with CRISPRi beyond a cell-free environment. In the field, researchers require a tool to detect the presence of CRISPRi’s effectiveness in vivo. However, integrating a detection system in the microbe proves to be no easy feat (Chen et al., 2022). Toeholds present a solution to this problem as we can use it as an independent biosensor system to detect the target gene’s presence in the microbe. There are two distinct scenarios that our toehold system will be able to detect:

1. Our CRISPRi system does not work and mRNA is produced.
2. Our CRISPRi system works and no mRNA is produced.

In the case that the CRISPRi sgRNA does not bind to the target DNA, mRNA complementary to our toehold will be produced. This would permit the binding of the target mRNA to the switch, allowing the elongation of the toehold, and revealing Green Fluorescent Protein (GFP) and producing fluorescence (see Fig. 1) . In the case that the CRISPRi system works as intended, the mRNA will not be produced and therefore no GFP would fluoresce in our toehold system (see Fig. 2). Due to CRISPRi’s ability to downregulate a target gene utilizing deactivated Cas9 proteins, toehold switches can provide insight to the degree of downregulation achieved. In the case that CRIPSRi successfully downregulates a target gene, no RNA will be produced thus no trigger will bind to the switch sequence of the toehold. Unsuccessful downregulation, on the other hand, would allow for RNA of a target gene to be transcribed thus providing the toehold switch with an activating trigger.

Figure 1. Graphical representation of how an active CRISPRi system works in conjunction with our toehold system when the CRISPRi system is active.

Figure 2. Graphical representation of how an inactive CRISPRi system works in conjunction with our toehold system.

Toehold biosensors allow for an inexpensive and highly specific means to gene quantification, as seen with toehold detection systems developed in 2019 for the SARS-CoV-2 virus (Geraldi et al., 2022). For all of these reasons, our team decided that toeholds would be the most viable detection system.

The integration of toehold biosensors with CRISPRi systems will significantly enhance researchers’ ability to quantify and monitor gene expression within living cells. This powerful combination will provide an accurate and sensitive method for measuring CRISPRi’s efficiency, enabling more precise optimization of gene silencing strategies and accelerating the development of CRISPRi-based therapeutics. For our purposes, our team decided to conduct experimentation within cell-free systems for ease of optimization of reaction dynamics. Furthermore, the adaptability of toehold biosensors allows for rapid customization to detect various target genes, making this approach a versatile tool for a wide range of CRISPRi applications in both basic research and clinical settings.

Methodology

Toehold switches are riboregulators that consist of a specific RNA switch site and a ribosomal binding site (RBS). These switches have a complementary trigger that “activates” the quantification system when present. Toehold switches utilize a special RNA sequence that forms a hairpin loop, preventing translation of the downstream reporter gene, which, in our case, is Green Fluorescent Protein (GFP). However, when the trigger binds to the switch sequence, the toehold structure elongates. This exposes the RBS, allowing for the translation of the reporter gene (Green et al., 2014).

This detection system quantifies our CRISPRi system’s ability to downregulate a target gene. Simply stated, when the target gene is present – the switch is activated. However, when the gene is successfully downregulated, the switch remains inactive, resulting in negligible GFP output (see Fig. 3).

Figure 3. Animation showing the activation and elongation of our toehold switch.

References